Silencing of Fem1cR3 gene expression in the DBA/2J mouse precedes retinal ganglion cell death and is associated with histone deacetylase activity

Abstract

Purpose: Downregulation of normal gene expression in dying retinal ganglion cells has been documented in both acute and chronic models of optic nerve disease. The authors examined the mechanism and timing of this phenomenon in DBA/2J mice, using genetically modified substrains of this inbred line.

Methods: DBA/2J mice, doubly congenic for the Bax mutant allele and the ganglion cell reporter gene Fem1c(Rosa3) (R3), were evaluated to elucidate the timing of loss of normal gene expression during the apoptotic process. The localization of histone deacetylase 3 (HDAC3) and nuclear histone H4 acetylation were examined by immunofluorescence in dying cells. The role of HDACs in gene silencing during glaucoma was interrogated using the global HDAC inhibitor trichostatin A (TSA).

Results: Silencing of the R3 allele occurred in Bax(-/-) ganglion cells, indicating that this process preceded the committed step of the intrinsic apoptotic pathway. Weekly TSA treatment, between the ages of 6 and 10 months, was able to attenuate the loss of R3 expression in the retina, but had no effect on optic nerve degeneration. Dying cells in aging DBA/2J mice exhibited nuclear localization of HDAC3 and a decrease in the level of H4 acetylation.

Conclusions: Retinal ganglion cells exhibit a loss of normal gene expression as an early (pre-BAX involvement) part of their apoptotic program during glaucomatous degeneration. This process can be ameliorated, but not completely blocked, using HDAC inhibitors. Epigenetic changes to active chromatin, such as deacetylation, may be mediated by HDAC3 in dying neurons.

Figure 1.

Scatterplot of total neuron counts relative to percentage of βGeo expression in aged DBA/2J^R3 mice with different Bax genotypes. Randomly selected 100 × 100-μm fields were evaluated in double-labeled retinal whole mounts of mice 10 months of age. Each field was also designated as one of three phenotypes based on the apparent percentage of cells expressing βGEO based on X-gal staining. Wild-type (red circles) and Bax^+/− (blue squares) mice exhibited similar cell densities for phenotype regions 1 and 2, but a lower density of cells in phenotype 3 regions. Bax^−/− mice (green triangles) exhibited approximately twice the density of cells, which did not decrease in regions with phenotype 3. See Table 1 for quantitative values and statistics.

Figure 2.

Regional loss of βGEO activity occurs in 10-month-old Bax^−/− DBA/2J^R3/R3 mice. Cells with βGEO activity appear blue, whereas Nissl-stain appears violet. (A) Whole-mounted retina, showing a wedge-shaped region devoid of βGEO activity (defined by white hashed lines). Regions (B) and (C) in borders of the wedge are shown in higher magnification. Although cells have stopped expressing βGEO, they are still present in the wedge region. Scale bar for (B) and (C): 50 μm.

Figure 3.

HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells, HDAC3, and counterstained for DNA using DAPI. (A) Merged image of all three labels in a representative section. (B–D) Individual channels for DAPI, HDAC3, and γH2AX. Based on the γH2AX labeling, the nuclei in the section were identified as stages I, II, or III. Stage II and stage III cells exhibit nuclear localization of HDAC3. Stage II and stage III cells also appear to contain fragmented or highly condensed chromatin, respectively. N, representative nucleus. Scale bar: 5 μm.

Figure 4.

Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells, acetylated histone H4 (AcH4), and counterstained for DNA using DAPI. (A) Merged image of all three labels in a section. (B–D) Individual channels for DAPI, AcH4, and γH2AX, respectively. Representative stages I, II, or III nuclei (N), based on the γH2AX labeling, are indicated. Stage I cells exhibit strong labeling for AcH4, whereas the single stage III cell in the image has virtually no AcH4 present. Stage II cells show some AcH4 staining, but it is typically intermediate between stage I and stage III labeling. Scale bar: 5 μm.

Figure 5.

TSA treatment attenuates the downregulation of Fem1c^R3 expression. Mice treated weekly with vehicle (DMSO), or TSA, between 6 and 10 months of age, were compared with age-matched mice that had no treatment (No). The level of Fem1c^R3 gene expression was assessed as a function of the amount of βGEO fusion protein activity relative to total protein in retinal homogenates. TSA provided a significant level (asterisk) of attenuation of the loss of βGEO enzyme activity observed in both noninjected and DMSO-injected mice (P = 0.032 and P = 0.049, respectively). The preservation of βGEO activity afforded by TSA is only partial, however, and represents approximately 80% of the level observed in young DBA/2J mice without disease.

Figure 6.

Optic nerve scores for treated 10-month-old mice. Optic nerves for each eye harvested for βGEO activity (Fig. 5) were also examined for optic nerve degeneration. Sections were immunostained for βIII tubulin and counterstained with DAPI. Digital images were scored by three masked observers for (A) mild, (B) moderate, and (C) severe damage, based on the extent of positive staining for βIII tubulin. The frequency of each category, for each treatment group, was then graphed and evaluated (D). TSA treatment provided no statistical difference in the frequencies of mild, moderate, or severe damage to the optic nerves of mice, compared with either noninjected mice (No), or vehicle-injected mice (DMSO) (χ² test, P = 0.051 and P = 0.207, respectively). Scale bar for (A–C): 100 μm.